Base sequence detecting electrode, base sequence detecting device and base sequence detecting method

ABSTRACT

A conductive detecting electrode ( 2 ), first blocking molecules ( 21 ) formed so as to cover a surface of the detecting electrode ( 2 ), the first blocking molecules decreasing adsorption of an intercalating agent to the surface of the detecting electrode ( 2 ), a target-complementary probe ( 23 ) immobilized to the detecting electrode ( 2 ) via a spacer member ( 22 ) comprising straight chain organic molecules, the target-complementary probe including a base sequence complementary to a target base sequence which is an object of detection, a conductive comparison electrode ( 3 ), and second blocking molecules ( 31 ) formed so as to cover a surface of the comparison electrode ( 3 ), the second blocking molecules decreasing adsorption of an intercalating agent to the surface of the comparison electrode ( 3 ), are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/239,175, filed Sep. 25, 2002, now allowed as U.S. Pat. No. 7,097,751B2, which is a 371 of PCT/JP02/08671, filed Aug. 28, 2002, and claimspriority to Japanese patent application No. 2002-244018, filed Aug. 23,2002, all of which are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a base sequence detecting electrode, abase sequence detecting device and a base sequence detecting method forspecifically detecting a specific gene existing in a sample.

BACKGROUND ART

Due to developments in genetic engineering in recent years, in themedical field, diagnosis and prevention of diseases by genes has come tobe possible. Diagnosis by using genetic engineering is called genediagnosis. In gene diagnosis, by detecting defect or change in a gene ofa human, which may be source of a disease, diagnosis or measurement canbe carried out before the onset of a disease or at the initial stagethereof. Further, by detecting a gene of a contracted virus orpathogenic bacteria, an accurate diagnosis is possible.

A general gene detecting method conventionally used is as follows.

First, a gene is extracted from a sample. If necessary, the gene is cutby appropriate restriction endonuclease, and thereafter, electrophoresisand Southern plotting are carried out. Next, a nuclease probe having abase sequence complementary to the target gene which is the object ofdetection is hybridized with the plotted gene. Note that the nucleaseprobe is usually labeled by a fluorescent dye. Then, the fluorescent dyeis excited by laser light. Accordingly, the hybridized nuclease probe isdetected, and the existence of the target gene is verified.

However, in this detecting method using a fluorescent dye, it takes atleast a few days until detection of a gene. Further, the nuclease probemust be labeled by a high-priced fluorescent dye. In addition, a lasergenerating device for exciting the fluorescent dye is needed, and thedevice becomes large.

In order to solve the above-described problems of the detecting methodusing a fluorescent dye, a gene detecting method using anelectrochemical method has been conceived of. The detecting method usingan electrochemical method is disclosed in Japanese Patent No. 2573443conceived of by the present inventors, and the contents thereof areincorporated herein by reference. In accordance with thiselectrochemical method, detecting an electrochemical signal from anelectrode having a probe immobilized thereto allows existence of thetarget gene to be verified.

However, in gene detection using an electro-chemical method, backgroundcurrent arises at the time of current detection. Accordingly, currentcaused by derivation with the probe and the background current areincluded in the current value detected from the electrode. Therefore,from the results of detection, it has been difficult to extract only thecurrent derived from the probe.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a base sequencedetecting electrode, a base sequence detecting device and a basesequence detecting method which precisely carry out detection of currentbased on an interaction with a target base sequence.

According to one aspect of the present invention, there is provided abase sequence detecting electrode comprising: a conductive detectingelectrode; first blocking molecules formed so as to cover a surface ofthe detecting electrode, the first blocking molecules decreasingadsorption of an intercalating agent to the surface of the detectingelectrode; a target-complementary probe immobilized to the detectingelectrode, the target-complementary probe having a base sequencecomplementary to a target base sequence which is an object of detection;a conductive comparison electrode; and second blocking molecules formedso as to cover a surface of the comparison electrode, the secondblocking molecules decreasing adsorption of an intercalating agent tothe surface of the comparison electrode.

According to another aspect of the present invention, there is provideda base sequence detecting device comprising: a conductive detectingelectrode; first blocking molecules formed so as to cover a surface ofthe detecting electrode, the first blocking molecules decreasingadsorption of an intercalating agent to the surface of the detectingelectrode; a target-complementary probe immobilized to the detectingelectrode, the target-complementary probe having a base sequencecomplementary to a target base sequence which is an object of detection;a conductive comparison electrode; second blocking molecules formed soas to cover a surface of the comparison electrode, the second blockingmolecules decreasing adsorption of an intercalating agent to the surfaceof the comparison electrode; and a subtracter which subtracts anelectrochemical signal detected at the comparison electrode from anelectrochemical signal detected at the detecting electrode.

According to still another aspect of the present invention, there isprovided a base sequence detecting method comprising: detectingelectrochemical signals at a detecting electrode and a comparisonelectrode of a base sequence detecting device comprising: the conductivedetecting electrode; first blocking molecules formed so as to cover asurface of the detecting electrode, the first blocking moleculesdecreasing adsorption of an intercalating agent to the surface of thedetecting electrode; a target-complementary probe immobilized to thedetecting electrode via a first spacer member comprising straight chainorganic molecules, the target-complementary probe having a base sequencecomplementary to a target base sequence which is an object of detection;the conductive comparison electrode; and second blocking moleculesformed so as to cover a surface of the comparison electrode, the secondblocking molecules decreasing adsorption of an intercalating agent tothe surface of the comparison electrode; and subtracting anelectrochemical signal detected at the comparison electrode from anelectrochemical signal detected at the detecting electrode.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram showing a concept of an entire structure of abase sequence detecting device according to an embodiment of the presentinvention.

FIG. 2A and FIG. 2B are diagrams showing detailed structures of adetecting electrode and a comparison electrode according to theembodiment.

FIG. 3 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 4 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 5 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 6 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 7 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 8 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 9 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 10 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 11 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 12 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 13 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 14 is a top view of an electrode structure including the detectingelectrode and the comparison electrode according to the embodiment.

FIG. 15 is a flowchart of operation of the base sequence detectingdevice according to the embodiment.

FIG. 16 is a diagram showing a modified example of the comparisonelectrode of the base sequence detecting device according to theembodiment.

FIG. 17 is a graph showing results of current measurement of aconventional base sequence detecting device.

FIG. 18 is a graph showing results of current measurement of the basesequence detecting device including the comparison electrode.

FIG. 19 is a graph showing results of current measurement of a basesequence detecting device in which ethylene glycol molecules are used asa spacer member.

FIG. 20 is a graph showing results of current measurement of a basesequence detecting device in which mercapto octanol is used as blockingmolecules.

FIG. 21 is a graph showing results of current measurement of a basesequence detecting device in which straight chain alkane molecules areused as a spacer member.

FIG. 22A and FIG. 22B are diagrams showing a modified example in which atarget nuclease probe is immobilized without using a spacer member.

BEST MODE FOR CARRYING OUT OF THE INVENTION

Hereinafter, embodiments of the present invention will be described withreference to the figures.

FIG. 1 is a block diagram showing a concept of an entire structure of abase sequence detecting device according to an embodiment of the presentinvention. As shown in FIG. 1, a detecting electrode 2 and a comparisonelectrode 3 are disposed in an electro-chemical cell 1. Electric currentat the detecting electrode 2 and comparison electrode 3 are respectivelydetected at potentiostats 40 and 50. The obtained electrochemicalsignals are respectively outputted to signal processing units 60 and 70.The signal processing units 60 and 70 carry out a signal processing suchas calculation of a peak current value or the like, and output thesignal-processed data to a subtracter 80.

The subtracter 80 subtracts the data obtained from the signal processingunit 70 from the data obtained from the signal processing unit 60, andoutputs the results of subtraction to a computer 90.

The electrochemical cell 1, the detecting electrode 2, the comparisonelectrode 3, and the potentiostats 40 and 50 are formed in and/or on asemiconductor substrate 101 (chip) for detecting a base sequence.Further, the detecting electrode 2, the comparison electrode 3, and thepotentiostats 40 and 50 are comprised of an integrated circuit of thesemiconductor chip. The subtracter 80 may be realized as an integratedcircuit formed in and/or on the semiconductor substrate 101, or may berealized by a computer or a subtracter provided separately from thesemiconductor substrate 101. The subtracter 80 may be realized by acomputer and software controlling the computer, or may be realized asfirmware in which the subtraction processing contents are recorded on aROM or the like, or may be realized by only hardware.

The computer 90 may be any of computers which enable general arithmeticprocessing, for example, a personal computer or the like. The computer90 comprises a communication interface 91, a CPU 92, and a displaydevice 93. The communication interface 91 receives the results ofsubtraction from the subtracter 80, and outputs them to the CPU 92. TheCPU 92 displays the results of subtraction on the display device 93.

FIG. 2A and FIG. 2B are diagrams showing detailed structures of thedetecting electrode 2 and the comparison electrode 3. FIG. 2A shows thedetails of the detecting electrode 2, and FIG. 2B shows the details ofthe comparison electrode 3.

As shown in FIG. 2A, blocking molecules 21 are formed so as to cover thesurface of the detecting electrode 2. The blocking molecule 21 iscomprised of a molecule whose adsorption of an intercalating agent perunit area is less than that at the surface of the detecting electrode 2.Specifically, the blocking molecule 21 is comprised of, for example,mercapto hexanol, mercapto octanol, or the like. It is possible for thedetecting electrode 2 to not be completely covered, and it suffices thatat least one portion of the surface of the detecting electrode 2 iscovered. In this way, by making a structure such that the blockingmolecules 21 cover the surface of the detecting electrode 2, adsorptionof the intercalating agent molecules to the surface of the detectingelectrode 2 can be decreased.

The blocking molecule 21 is not particularly limited to theabove-described materials. However, for example, straight chain alkane,alkene, alkyne, ether, ester, and ketones are preferable, and further,the blocking molecule 21 may be a molecule in which a plurality of thesemolecules are connected in a chain via atoms such as oxygen, nitrogen,sulphur, or the like. Further, molecules, in which a group which, among,for example, an alkyl group, a hydroxyl group, a carboxyl group, asulfonic group, a nitro group, a phenyl group, an amino group, a thiolgroup, a halogen, and the like, is hard to interact with theintercalating agent molecules is introduced as a functional group, aremore preferable. Further, it is preferable that these blocking molecules21 are made to adsorb to the surface of the detecting electrode 2 via athiol group, an amino group, or the like, and self-organizingmonomolecular film is prepared. Other than this, an inorganic oxidelayer, a macro-molecular layer, or the like may be formed.

Further, an end of a spacer member 22 is immobilized to the surface ofthe detecting electrode 2 on which the blocking molecules 21 are notformed. The spacer member 22 is formed from a material in whichadsorption of the intercalating agent per unit area is less than that ofthe surface of the detecting electrode 2. Specifically, the spacermember 22 is comprised of straight chain organic molecules, and forexample, ethylene glycol is appropriate. The material of the spacermember 22 is not particularly limited. However, for example, straightchain alkane, alkene, alkyne, ether, ester, and ketones are preferable.Further, the spacer member 22 may be a molecule in which a plurality ofthese molecules are connected in a chain via atoms such as oxygen,nitrogen, sulphur, or the like.

Moreover, a target-complementary nucleic acid probe 23 is immobilized tothe other end of the spacer member 22. The target-complementary nucleicacid probe 23 is a probe comprising a nucleic acid having a basesequence complementary to the target nucleic acid sequence which is theobject of detection. Thus, by using a structure in which thetarget-complementary nucleic acid probe 23 is immobilized to thedetecting electrode 2 via the spacer member 22, the efficiency of thebinding of the sample and the target-complementary nucleic acid probe 23can be improved.

Conventionally, the intercalating agent molecules interact with thespacer member, and it is a cause of increasing the background current.However, as in the present embodiment, by selecting molecules, which itis hard for the intercalating agent molecules to interact with, as thestructure of the spacer member 22, the background current can bedecreased. The background current indicates current which is a cause ofnoise or the like other than the current caused by interaction betweenthe target-complementary nucleic acid probe 23 disposed at the detectingelectrode 2 and the intercalating agent. The background current includescurrent caused by, for example, interaction between the surface of thedetecting electrode 2 and the intercalating agent, interaction betweenthe blocking molecules 21 and the intercalating agent, and interactionbetween the spacer member 22 and the intercalating agent. The currentcaused by interaction between the target-complementary nucleic acidprobe 23 and the intercalating agent is called probe current.

When the target-complementary nucleic acid probe 23 comprising straightchain organic molecules as described above is used, the backgroundcurrent is substantially only a current caused by the intercalatingagent interacting with the surface of the detecting electrode 2.Therefore, the comparison electrode 3 to be described later may becomprised of only a blocking molecule layer, and it is extremelydesirable. As a spacer molecule having such an effect, for example,there is a straight chain molecule to which one or a plurality ofethylene glycol are linked. Other than this, provided that the spacermolecule is a molecule which does not interact with the intercalatingagent molecule, the material thereof is not limited.

As shown in FIG. 2B, at the comparison electrode 3, blocking molecules31 are formed so as to cover the surface of the comparison electrode 3.The blocking molecule 31 is preferably comprised of the same material asthat of tile blocking molecules 21 covering the surface of the detectingelectrode 2. However, it is not limited to that as far as it is amolecule exhibiting similar properties. The blocking molecule 31 may notcompletely cover the comparison electrode 3, and it suffices for atleast one portion of the surface of the comparison electrode 3 to becovered. Thus, by using a structure in which the surface of thecomparison electrode 3 is covered with the blocking molecules 31 whosematerial is the same as that of the blocking molecules 21, adsorption ofthe intercalating agent molecules to the surface of the detectingelectrode 2 can be decreased, and the effect of reducing the effectswith respect to the background currents at the detecting electrode 2 andthe comparison electrode 3 can be the same.

FIG. 3 to FIG. 14 are schematic top views of electrode structuresincluding the detecting electrode 2 and the comparison electrode 3. Asshown in FIG. 3 to FIG. 14, as the types of electrodes, there are acounter electrode 4 and a reference electrode 5, in addition to thedetecting electrode 2 and the comparison electrode 3. These detectingelectrode 2, comparison electrode 3, counter electrode 4, and referenceelectrode 5 are formed from conductive materials, and these electrodes 2to 5 are disposed in the electrochemical cell 1. A sample having ananalyte base sequence for carrying out hybridization between the probesimmobilized to the detecting electrode 2 and the comparison electrode 3,an intercalating agent, a buffer, and the like are filled in theelectrochemical cell 1 in which these detecting electrode 2, comparisonelectrode 3, counter electrode 4, and reference electrode 5 aredisposed, and electrochemical reaction is carried out therein.

The detecting electrode 2 and the comparison electrode 3 are electrodesfor detecting the reaction current in the cell 1.

A DNA probe having a target-complementary base sequence which iscomplimentary to the target base sequence is immobilized to thedetecting electrode 2. Probe current caused by interaction between thetarget-complementary nucleic acid probe and the intercalating agent isdetected from the detecting electrode 2.

The comparison electrode 3 is an electrode which detects backgroundcurrent such as noise or the like, and thereafter, decreases the effectsof the noise or the like by subtracting the background current from thecurrent detected at the detecting electrode 2. The comparison electrode3 is used without the probe being not fixed, or is used with a dummyprobe being immobilized as shown in FIG. 16 described later.

The counter electrode 4 is an electrode supplying electric current tothe inside of the cell 1 by applying a predetermined voltage between thedetecting electrode 2 or the comparison electrode 3.

The reference electrode 5 is an electrode which negatively feeds backthe electrode voltage to the counter electrode 4 in order to control thevoltage between the reference electrode 5 and the detecting electrode 2,or the reference electrode 5 and the comparison electrode 3, to apredetermined voltage characteristic. The voltage by the counterelectrode 4 is controlled by the reference electrode 5, and oxidationcurrent detection, which does not depend on various types of detectingconditions in the cell 1 and is highly accurate, can be carried out.

FIG. 3 shows a detecting electrode side 3-electrode system 31 comprisingthe detecting electrode 2, the counter electrode 4, and the referenceelectrode 5, and a comparison electrode side 3-electrode system 32comprising the counter electrode 3, the counter electrode 4, and thereference electrode 5.

At the detecting electrode side 3-electrode system 31, the rectangularcounter electrodes 4, which have a longitudinal direction in apredetermined direction, are disposed at predetermined intervals withrespect to the circular detecting electrodes 2. Further, the rectangularreference electrodes 5, which have a longitudinal direction in adirection substantially perpendicular to the longitudinal direction ofthe counter electrodes 4, are disposed at predetermined intervals withrespect to the detecting electrodes 2. An example is shown in which theelectrodes are disposed such that the distance between the detectingelectrode 2 and the counter electrode 4 and the distance between thedetecting electrode 2 and the reference electrode 5 are substantiallythe same. However, it is not limited to this example, and the elementsmay be disposed at different distances.

The comparison electrode side 3-electrode system 32 is configured suchthat the detecting electrode 2 of the detecting electrode side3-electrode system 31 is replaced with the comparison electrode 3. These3-electrode systems 31 and 32 are made to be an electrode system of oneset, and the potentiostats 40 and 50 are connected thereto.

FIG. 4 is a more specific electrode structural example configured on thebasis of the electrode structure of FIG. 3. As shown in FIG. 4, the3-electrode systems 31 and 32, which are the same as those shown in FIG.3, are alternately disposed in a matrix manner. In FIG. 4, an example inwhich the same 3-electrode systems are aligned in a column direction anddifferent 3-electrode systems are alternately aligned in a row directionis shown. However, the same 3-electrode systems may be aligned in therow direction, and different 3-electrode systems may be alternatelyaligned in the column direction. Further, different 3-electrode systemsmay be alternately aligned in both the row and column directions. Inthis case, the common 3-electrode systems are disposed so as to bealigned in oblique directions of the matrix.

FIG. 5 shows another arrangement example of electrodes having the sameshape as in FIG. 3. The reference electrodes 5, whose longitudinaldirections are the same as those of the rectangular counter electrodes4, are disposed at equal intervals so as to sandwich the counterelectrodes 4. The comparison electrodes 3 are disposed between thecounter electrode 4 and one reference electrode 5, and further, thedetecting electrodes 2 are disposed at the side of the referenceelectrode 5 opposite to the side at which the comparison electrodes 3are provided. Further, the detecting electrodes 2 are disposed betweenthe counter electrodes 3 and the other reference electrode 5, andfurther, the comparison electrodes 3 are disposed at the side of thereference electrode 5 opposite to the side at which the comparisonelectrodes 3 are provided. The distance between the counter electrode 4or the reference electrode 5 and the detecting electrodes 2 or thecomparison electrodes 3 are equal intervals. Further, the detectingelectrodes 2 and the comparison electrodes 3 are disposed at positionswhich are symmetrical across the reference electrode 5. Moreover, thedetecting electrodes 2 and the comparison electrodes 3 are disposed atpositions symmetrical across the counter electrode 4 so as to sandwichthe counter electrode 4.

FIG. 6 is a more specific electrode structural example configured on thebasis of the electrode structure of FIG. 5. As shown in FIG. 6, it has asimilar electrode structure as that shown in FIG. 5. However, aplurality of the detecting electrodes 2 and the comparison electrodes 3are disposed at equal intervals along the longitudinal directions of thecounter electrode 4 and the reference electrodes 5, and theyrespectively form detecting electrode groups 20 and comparison electrodegroups 3. Further, the detecting electrodes 2 and the comparisonelectrodes 3 are disposed at positions symmetrical across the referenceelectrodes 5 or the counter electrodes 4. The detecting electrodes 2 andthe comparison electrodes 3 disposed at symmetrical positions areelectrodes which are objects of subtraction. Namely, the 3-electrodesystem is comprised of the detecting electrodes 2 or the comparisonelectrodes 3 disposed at the positions symmetrical with respect to thereference electrode 5, and the reference electrode 5, and further, thecounter electrode 4 provided so as to be near to the detectingelectrodes 2 or the comparison electrodes 3. The distances from eachdetecting electrode 2 to the counter electrode 4 and the referenceelectrode 5 are equal, and the distances from each comparison electrode3 to the counter electrode 4 and the reference electrode 5 are equal.The potentiostat 40 or 50 is connected to the 3-electrode system. Aplurality of electrode sets, which are comprised of one detectingelectrode group 20, one comparison electrode group 30, one counterelectrode 4, and one reference electrode 5, are disposed in a directiondifferent from the longitudinal direction of these electrodes orelectrode groups (preferably the substantially perpendicular direction).

FIG. 7 shows another arrangement example of electrodes having the sameshape as in FIG. 3 and FIG. 5. The reference electrode 5 is disposed ata predetermined interval from the vicinity of the center of the counterelectrode 4 and such that the longitudinal direction thereof issubstantially perpendicular to the counter electrode 4. The detectingelectrode 2 is disposed at a predetermined interval from the referenceelectrode 5, and the comparison electrode 3 is disposed at a positionsymmetrical to the detecting electrode 2 with respect to the referenceelectrode 5.

FIG. 8 is a more specific electrode structural example configured on thebasis of the electrode structure of FIG. 7. As shown in FIG. 8, it has asimilar electrode structure as that shown in FIG. 7. A plurality of thedetecting electrodes 2 and the comparison electrodes 3 are disposed atpositions which are symmetrical across the reference electrode 5 at aequal intervals along the longitudinal directions of the referenceelectrode 5, and respectively form the detecting electrode group 20 andthe comparison electrode group 30. The detecting electrodes 2 and thecomparison electrodes 3 disposed at symmetrical positions with respectto the reference electrode 5 are electrodes which are objects ofsubtraction. Namely, the 3-electrode system is comprised of thedetecting electrodes 2 or the comparison electrodes 3 disposed atpositions symmetrical with respect to the reference electrode 5, and thereference electrode 5, and further, the counter electrode 4 provided soas to be perpendicular to the reference electrode 5. The potentiostat 40or 50 is connected to the 3-electrode system. The distances from eachdetecting electrode 2 to the reference electrode 5 are equal, and thedistances from each comparison electrode 3 to the reference electrode 5are equal.

FIG. 9 shows an other arrangement example of electrodes having the sameshape as in FIGS. 3, 5 and 7. Reference electrodes 51, 52, which havelongitudinal directions substantially parallel to the longitudinaldirection of the counter electrode 4, are disposed at predeterminedintervals from the counter electrode 4. The detecting electrode 2 isdisposed between the one reference electrode 51 and the counterelectrode 4, and the comparison electrode 3 is disposed between theother reference electrode 52 and the counter electrode 4. The positionalrelationship of the reference electrode 51 for the detecting electrode 2with respect to the counter electrode 4, and the positional relationshipof the reference electrode 52 for the comparison electrode 3 withrespect to the counter electrode 4 are equal. A set of the detectingelectrode 2 and the reference electrode 51 provided in correspondencewith the detecting electrode 2 is called a detecting electrode sideelectrode group 510, and a set of the comparison electrode 3 and thereference electrode 52 provided in correspondence with the comparisonelectrode 3 is called a comparison electrode side electrode group 520. A3-electrode system is comprised of one detecting electrode sideelectrode group 510 and the counter electrode 4, and the potentiostat 40is connected thereto. A 3-electrode system is comprised of onecomparison electrode side electrode group 520 and the counter electrode4, and the potentiostat 50 is connected thereto.

FIG. 10 is a more specific electrode structural example configured onthe basis of the electrode structure of FIG. 9. As shown in FIG. 10, ithas a similar electrode structure as that shown in FIG. 9. It is amatrix structure in which a plurality of the detecting electrode sideelectrode groups 510 are disposed in the column direction at equalintervals, and a plurality of the comparison electrode side electrodegroups 520 are disposed in the row direction at equal intervals, and theboth electrode groups are alternately disposed in the row direction. The3-electrode system is comprised of each of electrode groups 510, 520,and the counter electrode 4, and the potentiostat 40 or 50 is connectedthereto. Note that an example is shown in which the same electrodegroups are disposed in the column direction. However, it may be astructure in which the detecting electrode side electrode groups 510 andthe comparison electrode side electrode groups 520 are alternatelydisposed in the column direction as well. Further, a case is shown inwhich the row direction and the column direction are substantiallyperpendicular to each other. However, it is not limited to this, and thecolumn direction may have an angle which is not perpendicular to the rowdirection.

FIG. 11 is a diagram showing an example of an electrode structureconfigured of the detecting electrode 2 and the comparison electrode 3having the same shape as in FIGS. 3, 5, 7, and 9, and the circularcounter electrode 4 and the ring-shape reference electrode 5. As shownin FIG. 11, the ring-shape reference electrode 5 having the same thecenter as the circular counter electrode 4 is disposed so as to surroundthe counter electrode 4. Moreover, the detecting electrode 2 and thecomparison electrode 3 are disposed at positions symmetrical withrespect to the counter electrode 4 and at a predetermined interval fromthe outer periphery of the reference electrode 5.

FIG. 12 is a more specific electrode structural example configured onthe basis of the electrode structure of FIG. 11. As shown in FIG. 12, ithas a similar electrode structure as that shown in FIG. 11, and aplurality of the detecting electrodes 2 and a plurality of thecomparison electrodes 3 are disposed at positions symmetrical withrespect to the counter electrode 4. Further, because each of theplurality of detecting electrodes 2 and each of the plurality ofdetecting electrodes 2 are disposed at the same position from thecounter electrode 4, it is configured such that the plurality ofdetecting electrodes 2 and counter electrodes 3 are alternately disposedon a circumference 400 whose center is the counter electrode 4. Both ofthe detecting electrode 2 and the comparison electrode 3 disposed at thepositions symmetrical with respect to the counter electrode 4 may beobjects of subtraction, or both of the detecting electrode 2 and thecomparison electrode 3 which are adjacent to each other may be objectsof subtraction.

FIG. 13 is a diagram showing an example of an electrode structureconfigured of the detecting electrodes 2 and the comparison electrodes 3having the same shape as in FIGS. 3, 5, 7, and 9, and the square counterelectrode 4 and the cross-shaped reference electrode 5. As shown in FIG.13, the cross-shaped reference electrode 5, which is configured suchthat two rectangular electrodes whose longitudinal directionssubstantially perpendicularly intersect each other overlap, is disposed.At each of the four regions partitioned by the cross of the referenceelectrode 5, one of the counter electrodes 4 is disposed at apredetermined interval from the reference electrode 5. Further, at tworegions among these four regions, one detecting electrode 2 is disposedrespectively, and one comparison electrode 3 is disposed at the othertwo regions respectively. These detecting electrodes 2 and comparisonelectrodes 3 are disposed at positions further apart from the referenceelectrode 5 than the counter electrodes 4. The distances from thereference electrode 5 to the respective counter electrodes 4 are equal,and the distances from the reference electrode 5 to the respectivedetecting electrodes 2 and the respective comparison electrodes 3 areequal.

FIG. 14 is a more specific electrode structural example configured onthe basis of the electrode structure of FIG. 13. As shown in FIG. 14,the structures of the counter electrode 4 and the reference electrode 5are common to those of FIG. 13. In the case of this FIG. 14, at each offour regions 401 through 404 partitioned by the reference electrode 5, aplurality of detecting electrodes 2 are disposed, or a plurality ofcomparison electrodes 3 are disposed. More specifically, a plurality ofdetecting electrodes 2 are disposed at the regions 401 and 404 which areadjacent to one another, and a plurality of comparison electrodes 3 aredisposed at the regions 402 and 403 which are adjacent to one another.Both of the detecting electrodes 2 and the comparison electrodes 3,which have the same interval from the reference electrode 5, are objectsof subtraction. Note that the detecting electrodes 2 may be disposed inat least one of the four regions 401 through 404, and the comparisonelectrodes 3 may be disposed in at least one region. Accordingly, thedetecting electrodes 2 may be disposed at three regions, or thedetecting electrodes 2 may be disposed at only one region. Further, acase is shown in which one counter electrode 4 for each region, namely,four counter electrodes 4 in total, are disposed. However, two or morecounter electrodes 4 may be disposed at each region. In addition, thedetecting electrodes 2 and the comparison electrodes 3 may be disposedso as to be mixed at one region.

The electrode arrangements of FIG. 3 to FIG. 14 shown above areexamples, and the shape, the size, the positional relationship, and thelike of each electrode can be varied. The electrode structures shown inFIG. 3 to FIG. 14 can be disposed at one semiconductor or a substrate ofglass or the like. Accordingly, the detecting electrodes 2 and thecomparison electrodes 3 are disposed in the same substrate. Thedetecting electrode 2 may be one or plural. The comparison electrode 3may be one or plural. The comparison electrode 3, which is an object ofsubtraction with respect to the detecting electrode 2, may be one orplural. Further the detecting electrode 2, which is an object ofsubtraction with respect to the comparison electrode 3, may be one orplural. Of course, the respective electrodes may be disposed on anothersubstrate.

Next, operation of the base sequence detecting device described abovewill be described along the flowchart of FIG. 15.

First, the detecting electrode 2 and the comparison electrode 3 aredisposed in the electro-chemical cell 1, and a sample (analyte solution)containing a nucleic acid which is the object of inspection is filled inthe cell 1. Further, the cell 1 is maintained at a predeterminedtemperature, and a hybridization reaction between the sample and atarget-complementary nucleic acid probe immobilized to the detectingelectrode 2 is promoted. After the hybridization reaction is completed,the sample is sent out from the interior of the cell 1, and after it isfilled with a buffer agent, the interior of the cell 1 is filled with anintercalating agent. A predetermined voltage is applied, under thefeedback of voltage by the reference electrode 5, between the detectingelectrode 2 and the comparison electrode 3 in the cell 1 in which theintercalating agent is filled and the counter electrode 4. Thus,electrochemical measurement is carried out by the potentiostats 40 and50 in parallel at the detecting electrode 2 and the comparison electrode3 (s1).

Next, peak current values of the current values from the detectingelectrode 2 and the comparison electrode 3 obtained by theelectrochemical measurement are detected (s2). The detection of the peakcurrent value is carried out in parallel at both of the detectingelectrode 2 and the comparison electrode 3. The detection of the peakcurrent values is executed at signal processing units 60 and 70. Thesignal processing units 60 and 70 extract the peak values of the currentwaveforms obtained from the potentiostats 40 and 50 or of the voltagewaveforms in which the current waveforms are current-voltage converted.Note that the obtained peak current values may be A/D converted, or maybe obtained after carrying out a statistical processing such as a meanvalue calculation of a plurality of detecting electrodes 2 or aplurality of comparison electrodes 3, or the like. The obtained peakcurrent values are outputted to the subtracter 80.

The subtracter 80 subtracts the peak current value for the comparisonelectrode 3 from the peak current value for the detecting electrode 2(s3). The peak current value for the detecting electrode 2 is obtainedon the basis of a current in which a probe current caused by theintercalating agent molecules interacting with the target-complementarynucleic acid probe and a background current detected as unintended noiseare added. In the background current, a current value caused by theintercalating agent molecules interacting with the surface of thedetecting electrode 2, and a current value caused by the intercalatingagent molecules interacting with the spacer member 22 connecting thenucleic acid probe portion and the surface of the substrate, areincluded.

The peak current value for the comparison electrode 3 is obtained on thebasis of background current detected as unintended noise. Accordingly,by subtraction, the current value due to the background current can besubtracted, and the current due to the probe can be detected.

The subtracter 80 outputs the results of subtraction to the computer 90.The computer 90 displays the results of subtraction on the displaydevice 93. An operator can specify the base sequence included in thesample by the displayed the results of subtraction. Of course, there isprovided a determining circuit which carries out determination on thepresence/absence of a base sequence included in the sample or the likeon the basis of the results of subtraction, and the results ofdetermination may be displayed on the display device 93.

In this way, in accordance with the present embodiment, in addition to aconventional gene detecting electrode as the detecting electrode 2, anelectrode which can detect only background current is provided as thecomparison electrode 3. After current values at the both electrodes 2, 3are detected, the current value of the comparison electrode 3 issubtracted from the current value of the detecting electrode 2 bycomputing on a integrated circuit on the substrate, and it is possibleto detect only the current value derived from the target gene from whichbackground current is removed. Because the blocking molecules are formedso as to cover the surfaces of the detecting electrode 2 and thecomparison electrode 3, effects of the background current due tointeraction of the surfaces of the electrodes and the intercalatingagent can be decreased. Further, by using straight chain organicmolecules as the spacer member, the background current caused by thespacer can be greatly decreased. Accordingly, even if a probe is notprovided at the comparison electrode 3, hardly any difference in thebackground current due to the presence/absence of a spacer arises.Accordingly, the trouble of immobilizing the spacer and the probe can beeliminated.

In a conventional current detecting type gene detecting method, it wasdifficult to extract only the current value derived from the target geneand to analyze it. In particular, at the time of discriminating a singlebase polymorphic, the current value increased due to a nonspecificallyhybridized gene, and it was difficult to discriminate it from thebackground current. However, in accordance with the present embodiment,such a problem can be solved.

The present invention is not limited to the above-described embodiments.

As the probe immobilized to the detecting electrode 2, a probe having atarget-complementary base sequence, which is a base sequencecomplementary with the target base sequence, is used. However, it is notlimited to this. For example, a probe (hereinafter called a targetsemi-complementary nucleic acid probe) may be used, which has a singlebase or several base sequences different from the target-complementarybase sequence and in which the complementation with the target basesequence is lower than that of the target-complementary base sequence.Further, both a target-complementary nucleic acid probe and a targetsemi-complementary nucleic acid probe may be immobilized to thedetecting electrode and used.

Further, in the above-described embodiment, an example is shown in whichthe surface of the comparison electrode 3 is covered with the blockingmolecules 31 and the spacer and the probe are not provided. However, itis not limited to this. FIG. 16 is a diagram showing a modified exampleof the comparison electrode 3 shown in FIG. 2B. At a comparisonelectrode 300 according to the modified example, an end of a spacermember 301 comprised of straight chain organic molecules is immobilizedvia blocking molecules 31. The spacer member 301 is comprised of thesame material as that of the spacer member 22 provided at the detectingelectrode 2.

A dummy probe 302 is immobilized to the other end of the spacer member301. The dummy probe 302 is a probe comprising nucleic acid which doesnot have a base sequence complementary to the target nucleic acidsequence and has a noncomplementary base sequence.

In this way, the probe can also be immobilized to the comparisonelectrode 3 side via the spacer. In accordance therewith, backgroundcurrent caused by the spacer member 22 at the detecting electrode 2 sidecan be subtracted by the current at the comparison electrode 3 side.

Further, an example is shown in which, at the detecting electrode 2side, the target-complementary nucleic acid probe 23 is immobilized tothe detecting electrode 2 via the spacer member 22. However, it is notlimited to this. For example, as shown in FIG. 22A and FIG. 22B, thetarget-complementary nucleic acid probe 23 may be immobilized to thedetecting electrode 2 not via the spacer member 22.

Although not particularly shown in FIG. 1, the computer 90 mayautomatically control the respective circuits formed on thesemiconductor substrate 101. In this case, the computer 90 outputscontrol instructions for controlling operations of the potentiostats 40,50, the signal processing units 60, 70, and the subtracter 80 via acommunication interface 91. The respective circuits operate on the basisof the received control instructions, and transmit the results ofoperation or the results of computation to the computer 90. Inaccordance therewith, detection of a base sequence can be automated.Further, in FIG. 1, there is shown a case in which the signal processingunits 60, 70 and the subtracter 80 are provided at the semiconductorsubstrate 101. However, the same functions as these may be realized atthe computer 90 side. In this case, the output signals of thepotentiostats 40 and 50 are transmitted to the computer 90, and signalprocessing and subtraction processing are executed at the computer 90side.

As described above, the present invention can precisely carry outdetection of current based on interaction with a target base sequence.

EXAMPLES

Hereinafter, more specific Examples of the base sequence detectingdevice in accordance with the present invention will be described.

In these Examples, the Example of the above-described present embodimentand a Conventional Example for comparing with the Example will bedescribed.

Conventional Example

In this Conventional Example, detection of a nucleic acid was carriedout without using the comparison electrode 3. As the detectingelectrodes 2, three Au electrodes of detecting electrodes 201, 202, and203 were used. As the sample nucleic acid, the promoter region of MxAprotein having SEQ ID No: 1 was used.

(1) Immobilization of Nucleic Acid Probe to Surface of Au Electrode

The aforementioned sample nucleic acid is the target nucleic acid. Thedetecting electrode 201 is immersed in a solution containing 10 μM of asingle chain nucleic acid probe as a complementary sequence 2 which iscomplementary to the probe having SEQ ID No: 1 of the target nucleicacid for one hour. In accordance therewith, immobilization of atarget-complementary nucleic acid probe 211 to the detecting electrode201 was carried out. In the same way, single chain nucleic acid probes(hereinafter called target semi-complementary nucleic acid probes 212,213) having sequences 3, 4 different by a single base from the basesequence complementary to the target nucleic acid, were respectivelyimmobilized to the detecting electrodes 202 and 203. These single chainnucleic acid probes 211 to 213 are respectively immobilized to thedetecting electrodes 201 to 203 via a spacer comprising 20 bases(cytosine). Next, the detecting electrodes 201 to 203 were immersed in 1mM of a mercapto hexanol aqueous solution, and thereby the portions inwhich the target-complementary nucleic acid probe 211 or the targetsemi-complementary nucleic acid probes 212 and 213 were not immobilizedwas blocked.

(2) Detection of Sample Nucleic Acid by Using the Nucleic Acid ProbeImmobilized Surface

The sample nucleic acid was amplified by PCR after being extracted. Thedetecting electrodes 201 to 203 prepared in (1) were immersed in 2×SSCsolution containing the sample nucleic acid and incubated for 60 minutesat 35° C., and thereby an annealing reaction was carried out.Thereafter, cleaning was carried out with 0.2×SSC solution. Moreover,after the detecting electrodes 201 to 203 and the comparison electrode 3were immersed for 15 minutes in solution containing 50 μM of Hoechst33258 solution which was the intercalating agent, the oxidation currentresponse of the Hoechst 33258 molecules was measured. The results of thecurrent measurement are shown in FIG. 17. As compared with the detectingelectrode 201, the current values of the detecting electrodes 202 and203 are low, however, it cannot be distinguished from backgroundcurrent.

Example 1

In this Example 1, detection of a nucleic acid in the same way as in theConventional Example was carried out by using the comparison electrode3. As the detecting electrodes 2, three Au electrodes, i.e., detectingelectrodes 201, 202, and 203, were used. As the sample nucleic acid, thepromoter region of MxA protein having SEQ ID No: 1 was used.

(1) Immobilization of Nucleic Acid Probe to Surface of Au Electrode

The aforementioned sample nucleic acid is the target nucleic acid. Thedetecting electrode 201 was immersed for one hour in a solutioncontaining 10 μM of a single chain nucleic acid probe having sequence 2complementary with the target nucleic acid, and thereby immobilizationof the target-complementary nucleic acid probe 211 was carried out. Inthe same way, single chain target semi-complementary nucleic acid probeshaving sequences 3, 4 different by one base from sequence 2, wererespectively immobilized to detecting electrodes 202 and 203. Moreover,a single chain nucleic acid probe (hereinafter called targetsemi-complementary nucleic acid probe) having sequence 5 which isnoncomplementary to sequence 2, was immobilized to the comparisonelectrode 3. The single chain nucleic acid probes are immobilized to theelectrodes via a spacer member comprising 20 bases (cytosine). Next, thedetecting electrodes 201 to 203 and the comparison electrode 3 wereimmersed in a 1 mM mercapto hexanol aqueous solution, and then theportions where the nucleic acid probes were not immobilized wereblocked.

(2) Detection of Sample Nucleic Acid by Using Nucleic Acid ProbeImmobilized Surface

The sample nucleic acid was amplified by PCR after being extracted. Thedetecting electrodes 201 to 203 and the comparison electrode 3 preparedin (1) were immersed in 2×SSC solution containing the sample nucleicacid and incubated at 35° C. for 60 minutes, and thereby an annealingreaction was carried out. Thereafter, cleaning was carried out with0.2×SSC solution. Moreover, after the detecting electrodes 201 to 203and the comparison electrode 3 were immersed for 15 minutes in asolution containing 50 μM of Hoechst 33258 solution which is theintercalating agent, the oxidation current response of the Hoechst 33258molecules was measured. After the current measurement, the backgroundcurrent was removed by a subtracter. The results after removal are shownin FIG. 18. It is shown that, at the detecting electrode 202, there ishardly any current value derived from the gene which is the object ofdetection, and the difference of the one base can be clearlydistinguished. On the other hand, it is found that, at the detectingelectrode 203, the gene which is the object is non-specificallyhybridized. In accordance with this Example 1, it was clear that onlythe current derived from the object gene can be extracted by carryingout gene detection by using the comparison electrode 3.

Example 2

In Conventional Example 1 and Example 1, as the spacer member of thenucleic acid probe, a base sequence which is easy to interact withintercalating agent molecules was used. Therefore, the nucleic acidprobe was immobilized to the comparison electrode 3 also. In thisExample 2, as the spacer member of the nucleic acid probe, ethyleneglycol molecules, which do not interact with the intercalating agentmolecules, were used. As the sample nucleic acid, the promoter region ofMxA protein having SEQ ID No: 1 was used.

(1) Immobilization of Nucleic Acid Probe to Surface of Au Electrode

The aforementioned sample nucleic acid is the target nucleic acid. Thedetecting electrode 201 was immersed for one hour in a solutioncontaining 10 μM of a single chain complementary nucleic acid probe 211having sequence 2 complementary with the target nucleic acid, andthereby immobilization of the nucleic acid probe was carried out. In thesame way, single chain target semi-complementary nucleic acid probes 212and 213 having sequences 3, 4 different by one base from the targetnucleic acid were immobilized to detecting electrodes 202 and 203.Immobilization of a single chain nucleic acid probe to the comparisonelectrode 3 is not carried out. The single chain nucleic acid probes 211to 213 were fixed to the respective electrodes via a spacer comprising30 ethylene glycol molecules. Next, the detecting electrodes 201 to 203and the comparison electrode 3 were immersed in a 1 mM mercapto hexanolaqueous solution, and then the portions where the nucleic acid probeswere not immobilized were blocked.

(2) Detection of Sample Nucleic Acid by Using Nucleic Acid ProbesImmobilized Surface

The sample nucleic acid was amplified by PCR after being extracted. Thedetecting electrodes 201, 202 and the comparison electrode 3 prepared in(1) were immersed in 2×SSC solution containing the sample nucleic acidand incubated at 35° C. for 60 minutes, an annealing reaction wascarried out. Thereafter, cleaning was carried out with 0.2×SSC solution.Moreover, after the detecting electrodes 201, 202 and the comparisonelectrode 3 were immersed for 15 minutes in a solution containing 50 μMof Hoechst 33258 solution which is the intercalating agent, theoxidation current response of the Hoechst 33258 molecules was measured.After the current measurement, the background current was removed by asubtracter. The results after the removal are shown in FIG. 19. It isshown that, at the detecting electrode 202, there is hardly any currentvalue derived from the gene which is the object of detection, and thedifference of the one base can be clearly distinguished. On the otherhand, it is found that, at the detecting electrode 203, the gene whichis the object is non-specifically hybridized. In accordance with thisExample 2, it was clear that, by using ethylene glycol as the spacer ofthe nucleic acid probe immobilized to the detecting electrode 2, itcould be comprised of only by blocking molecules without immobilizing anucleic acid probe to the comparison electrode 3.

Example 3

In this Example 3, blocking molecules other than those of theConventional Example and Examples 1 and 2 were used. As the spacermember of the nucleic acid probe, a molecule in which five ethyleneglycol molecules were bound was used. As the sample nucleic acid, thepromoter region of MxA protein having SEQ ID No: 1 was used.

(1) Immobilization of Nucleic Acid Probe to Surface of Au Electrode

The aforementioned sample nucleic acid is a target nucleic acid. Thedetecting electrode 201 was immersed for one hour in a solutioncontaining 10 μM of a single chain target-complementary nucleic acidprobe 211 having sequence 2 complementary with the sample nucleic acid,and thereby immobilization of the nucleic acid probe was carried out. Inthe same way, single chain target semi-complementary nucleic acid probes212 and 213 having sequences 3, 4 different by one base from the samplenucleic acid were immobilized to the detecting electrodes 202 and 203.Fixing of the single chain nucleic acid probe to the comparisonelectrode 3 was not carried out. The single chain nucleic acid probes211 to 213 were immobilized to the electrodes via a spacer comprising 30ethylene glycol molecules. Next, the detecting electrodes 201 to 203 andthe comparison electrode 3 were immersed in 1 mM of a mercapto octanolaqueous solution, and then the portions where the nucleic acid probeswere not fixed were blocked.

(2) Detection of Sample Nucleic Acid by Using the Nucleic Acid ProbeImmobilized Surface.

The sample nucleic acid was amplified by PCR after being extracted. Thedetecting electrodes 201 to 203 and the comparison electrode 3 preparedin (1) were immersed in 2×SSC solution containing the sample nucleicacid and incubated at 35° C. for 60 minutes, an annealing reaction wascarried out. Thereafter, cleaning was carried out with 0.2×SSC solution.Moreover, after the detecting electrodes 201 to 203 and the comparisonelectrode 3 were immersed for 15 minutes in a solution containing 50 μMof Hoechst 33258 solution which is the intercalating agent, theoxidation current response of the Hoechst 33258 molecules was measured.After the current measurement, the background current was removed by asubtracter. The results after removal are shown in FIG. 20. It is shownthat, at the detecting electrode 202, there is hardly any current valuederived from the gene which is the object of detection, and thedifference of the one base can be clearly distinguished. On the otherhand, it is found that, at the detecting electrode 203, the object geneis non-specifically hybridized.

Example 4

In this Example 4, as the spacer member of the nucleic acid probe,straight chain alkane molecules were used. As the sample nucleic acid,the promoter region of MxA protein having SEQ ID No: 1 was used.

(1) Immobilization of Nucleic Acid Probe to Surface of Au Electrode

The aforementioned sample nucleic acid is the target nucleic acid. Thedetecting electrode 201 was immersed for one hour in a solutioncontaining 10 μM of the single chain target-complementary nucleic acidprobe 211 having sequence 2 complementary with the sample nucleic acid,and thereby immobilization of the nucleic acid probe 211 to thedetecting electrode 201 was carried out. In the same way, the singlechain target semi-complementary nucleic acid probes 212 and 213 havingsequences 3, 4 different by one base from the sample nucleic acid wereimmobilized to the detecting electrodes 202 and 203. Fixing of a singlechain nucleic acid probe to the comparison electrode 3 is not carriedout. The single chain complementary nucleic acid probe 211 and thetarget semi-complementary nucleic acid probes 212 and 213 were fixed tothe electrodes via a spacer comprising straight chain alkane moleculeshaving 96 carbons. Next, the detecting electrodes 201 to 203 and thecomparison electrode 3 were immersed in a 1 mM mercapto hexanol aqueoussolution, and then the portions where the nucleic acid probes were notimmobilized were blocked.

(2) Detection of Sample Nucleic Acid by Using the Nucleic Acid ProbesImmobilized Surface

The sample nucleic acid was amplified by PCR after being extracted. Thedetecting electrodes 201 to 203 and the comparison electrode 3 preparedin (1) were immersed in 2×SSC solution containing the sample nucleicacid and incubated at 35° C. for 60 minutes, and thereby an annealingreaction was carried out. Thereafter, cleaning was carried out with0.2×SSC solution. Moreover, after the detecting electrodes 201 to 203and the comparison electrode 3 were immersed for 15 minutes in asolution containing 50 μM of Hoechst 33258 solution which is theintercalating agent, the oxidation current response of the Hoechst 33258molecules was measured. After the current measurement, the backgroundcurrent was removed by a subtracter. The results after removal are shownin FIG. 21. It is shown that, at the detecting electrode 202, there ishardly current value derived from the gene which is the object ofdetection, and the difference of the one base can be clearlydistinguished. In accordance with the Example 4, it was clear that, byusing straight chain alkane molecules as the spacer of the nucleic acidprobe, it can be configured by only blocking molecules withoutimmobilizing a nucleic acid probe to the comparison electrode 3.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

INDUSTRIAL APPLICABILITY

As described above, the present invention is advantageous in thetechnical field of detecting electrodes for detecting base sequences,the technical field of detecting devices for detecting base sequences,and the technical field of detecting methods for detecting basesequences.

1. A base sequence detecting method comprising: detectingelectrochemical signals at a detecting electrode and a comparisonelectrode of a base sequence detecting device comprising: the conductivedetecting electrode; first blocking molecules formed so as to cover asurface of the detecting electrode, the first blocking moleculesdecreasing adsorption of an intercalating agent to the surface of thedetecting electrode; a target-complementary probe immobilized to thedetecting electrode via a first spacer member comprising straight chainorganic molecules, the target-complementary probe including a basesequence complementary to a target base sequence which is an object ofdetection; the conductive comparison electrode; and second blockingmolecules formed so as to cover a surface of the comparison electrode,the second blocking molecules decreasing adsorption of an intercalatingagent to the surface of the comparison electrode; and subtracting anelectrochemical signal detected at the comparison electrode from anelectrochemical signal detected at the detecting electrode, wherein thebase sequence detecting device further comprising a dummy probeimmobilized to the comparison electrode via a second spacer membercomprising straight chain organic molecules, the dummy probe including abase sequence noncomplementary to the target base sequence which is theobject of detection, and wherein the detecting electrode is configuredto detect a probe current caused by interaction between theintercalating agent and the target-complementary probe hybridized withthe target, the comparison electrode is configured to detect backgroundcurrent.
 2. The base sequence detecting method according to claim 1,wherein the target-complementary probe is fixed to the detectingelectrode via the first spacer member comprising straight chain organicmolecules.
 3. The base sequence detecting method according to claim 1,wherein results of subtraction are displayed on a display deviceprovided at a computer.
 4. The base sequence detecting method accordingto claim 1, wherein the base sequence detecting device further comprisesa counter electrode which applies a predetermined voltage between thecounter electrode and the detecting electrode; and a first referenceelectrode to set voltage to be detected to the counter electrode.
 5. Thebase sequence detecting method according to claim 4, wherein the counterelectrode to apply a predetermined voltage between the counter electrodeand the detecting electrode, and between the counter electrode and thecomparison electrode.
 6. The base sequence detecting method according toclaim 1, wherein the first blocking molecules adsorb to the surface ofthe detecting electrode via one of a thiol group and an amino group toprepare an amino group self-organizing monomolecular film.
 7. The basesequence detecting method according to claim 1, wherein the straightchain organic molecules include one of straight chain alkane, alkene,alkyne, ether, ester, and ketones, and include a molecule in whichseveral molecules of straight chain alkane, alkene, alkyne, ether,ester, and ketones are connected in a chain via atoms including one ofoxygen, nitrogen, and sulphur.